Gaylord J. Knutson

Prognostic value of discs large homolog 7 transcript levels in prostate cancer.

Hypoxia has been associated with malignant progression, metastasis and resistance to therapy. Hence, we studied expression of hypoxia–regulated genes in 100 prostate cancer (CaP) bulk tissues and 71 adjacent benign tissues. We found 24 transcripts significantly overexpressed (p≤0.02). Importantly, higher transcript levels of disc large (drosophila) homolog-associated protein 5 (DLGAP5)/discs large homolog 7 (DLG7)/hepatoma up-regulated protein (HURP), hyaluronan-mediated motility receptor (HMMR) and cyclin B1 (CCNB1) were associated with higher Gleason score and more advanced systemic progression. Since the products of HMMR and CCNB1 have been identified recently as molecular markers of CaP progression, we postulated that DLG7 has prognostic value too. To test this hypothesis, we measured transcript levels for DLG7 in a 150-pair case-control cohort. The cases (progression to systemic disease within six years of surgery) and controls (no progression within eight years) were matched for clinical and pathologic prognostic variables, including grade, stage, and preoperative serum levels of PSA. The overall prognostic ability of DLG7, as tested in receiver operating characteristic analysis was of 0.74 (95% CI, 0.68 to 0.8). Overall, our data indicate that expression of DLG7, a hypoxia-controlled gene, holds prognostic potential in high-risk CaP; this also demonstrates that variation of oxygen tension may constitute a tool for identification of novel biomarkers for CaP.

Age-dependent response of murine female bone marrow cells to hyperbaric oxygen

Consequences of age on the effects of hyperbaric oxygen (HBO) on bone marrow (BM) derived stem cells and progenitors (SCPs) are largely unknown. We treated 2- and 18-month old C57BL/6 female mice by HBO. Hematopoietic stem cells and progenitors, enumerated as colony-forming units in culture, were doubled only in peripheral leukocytes and BM cells of young mice receiving HBO. In old mice colony-forming unit fibroblast numbers, a measure of mesenchymal stromal cells (MSCs) from BM, were high but unaffected by HBO. To further explore this finding, in BM-MSCs we quantified the transcripts of adipocyte early-differentiation genes peroxisome proliferator-activated receptor-γ, CCAAT/enhancer binding protein-β and fatty-acid binding protein 4; these transcripts were not affected by age or HBO. However, osteoblast gene transcripts runt-related transcription factor 2, osterix (OSX) and alkaline phosphatase (AP) were twofold to 20-fold more abundant in MSCs from old control mice relative to those of young control mice. HBO affected expression of osteoblast markers only in old MSCs (OSX gene expression was reduced by twofold and AP expression was increased threefold). Our data demonstrate the impact of aging on the response of BM SCPs to HBO and indicate the potentially different age-related benefit of HBO in wound healing and tissue remodeling.

Abstract C223: Targeting hypoxia for more effective immunotherapy of prostate cancer.

In early clinical trials, vaccination with allogeneic cancer cells enhanced the overall survival in some patients with malignancies. Because the administered vaccine cells were cultured at pO2=20 kPa, it is unclear whether they provided an adequate antigen match to tumor cells in situ where pO2 is generally much lower. Thus, we postulate that hypoxically grown vaccine cells will provide a better antigen match to tumors in situ. We are testing this hypothesis by studying the effects of hypoxia on prostate cancer (CaP) cells. LnCaP and VCaP cells were cultured at pO2=2 kPa or 20 kPa and analyzed by 2-D gel electrophoresis. The difference in pO2 affected the proteome mostly quantitatively (i.e., by change in spot intensity). Using a threshold of fivefold change, we found that hypoxia decreased the levels of thirteen proteins and increased of four in VCaP cells. These results are in line with the reports showing that hypoxia affects expression only of a small fraction of total cellular protein and that the content of total protein is not altered significantly. To determine if the hypoxia-dependent changes affected immune reactivity of the cells, we took advantage of the spontaneous autoantibodies against tumor-associated antigens (TAAs). Hence, we compared the binding to lysates of LnCaP and VCaP cells of antibodies pooled from CaP patients (n=25), healthy controls (n=25), rheumatoid arthritis (n=17), colorectal cancer (n=10) and lung cancer (n=10). CaP patient sera plasma reacted with numerous spots, some even in control groups and thus considered nonspecific. CaP sera plasma specifically bound to seven spots; four were hypoxia-specific. All selected spots were excised from the gel, trypsin-digested, and identified by MALDI-TOF mass spectrometry as heat shock 70 kDa protein 4; protein disulfide isomerase A3; heterogeneous nuclear ribonucleoprotein L and leucine-rich repeat-containing protein 47. With the exception of the latter, the proteins have been identified or validated as TAAs before, but apparently none in CaP. CaP patient antibodies bound to lysates of LnCaP cells grown at pO2=2 kPa, but less than to such VCaP cell lysates. Our results confirmed the findings that hypoxia affects the proteome mostly quantitatively. Lysates of hypoxic CaP cells revealed novel potential TAAs suggesting the potential immunologic relevance of antigens in the hypoxic proteome. Thus, pO2 control can provide a tool for the development of more effective immunotherapy for CaP and possibly other cancers. Support: DOD PC094680 (CRG), PCF Creativity Award (CRG), Mayo Clinic Prostate SPORE 5P50CA091956 (SV-P); Mrs. Adelyn L. Luther, Singer Island, Florida and Mayo Clinic Cancer Center (SV-P). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C223.

Abstract LB-308: Hypoxia affects gene expression and proteome of prostate cancer cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Tumor-associated hypoxia facilitates malignant progression, metastasis and poor prognosis in prostate cancer (CaP); however, effects of oxygen tension ( p O2) in CaP are just beginning to be investigated. To establish mechanisms whereby hypoxia enhances malignant properties and survival of CaP and to identify p O2-regulated tumor-associated macromolecules, we propagated CaP cell lines LnCaP, VCaP and DU145 in hypoxia ( p O2=2 kPa) and compared them with normoxically grown cells ( p O2=20 kPa); hypoxic cells proliferated faster and secreted more vascular endothelial growth factor ( p <0.05). Transcriptome studies revealed different gene expression in cells grown in hypoxia relative to those in normoxia. Interestingly, in hypoxic cells transcripts associated with cancer and urologic disease were overexpressed in comparison to normoxic cells ( p <0.001) suggesting an association of low p O2 and aggressive features of CaP. Further, we analyzed the transcriptome in primary CaP cells isolated by laser-capture microdissection (LCM) and whole CaP tissue. We compared the data to those from CaP cells cultured at p O2=20 kPa or p O2=2 kPa. Notably, hypoxia increased transcript levels for pyruvate dehydrogenase kinase isozyme 1, nuclear prelamin A recognition factor, glucose phosphate isomerase, and glyceraldehyde-3-phosphate dehydrogenase in all three cell lines ( p <0.05) to levels comparable to those found in primary bulk tissue and LCM isolated cells ( p <0.005). This finding suggests that gene expression in hypoxically cultured cells is more akin to that in tumor cells in situ than are cells grown normoxically. By 2D-gel electrophoresis, we found that change in p O2 affected the proteome mostly quantitatively ( i.e. , by change in spot intensity). Our results suggest that hypoxia affects transcriptome and proteome in CaP cells. In addition, we screened 20 patient sera and 20 healthy control sera for spontaneous antibodies cross-reactive with VCaP cells and found that the sera reacted with numerous proteins, some previously reported to elicit an immune response in CaP patients ( e.g. , nucleoporin 62 and transitional endoplasmic reticulum ATPase). By this method we detected numerous novel autoantigens (under validation). Currently we are clarifying the p O2 effects on the relationship of transcriptome, proteome and tumor-associated antigens in CaP cells. Support: DOD PC094680 (CRG), Minnesota Partnership for Biotechnology and Medical Genomics, Mayo Clinic Prostate SPORE 5P50CA091956 (FK); Mrs. Adelyn L. Luther, Singer Island, Florida and Mayo Clinic Cancer Center (SV-P). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-308. doi:10.1158/1538-7445.AM2011-LB-308